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Figures

Plate transfer assay for spore transfer. Shown is the transfer apparatus used to assess spore transfer from stainless steel (38). (A) Apparatus set up for spore transfer. The plunger is attached to a supporting metal frame. To maintain a sterile environment, a Bunsen burner is placed on either side of an electronic balance with a BHI agar plate on it. The balance was tared with each agar plate, and the spores plunged at a force of 100 g/10 s. (B) Inoculated stainless steel disc fastened to the metal plunger. (C) BHI agar plate compressed aseptically with the inoculated steel disc.

Effect of medium composition on spore formation. The abilities of eight clinical isolates of C. difficile to form spores in Columbia or BHI broth were determined. To determine the number of spores formed in each medium, viable counts were performed on the agar form of the medium in the presence and absence of 1% sodium taurocholate. A significant difference in the abilities of all strains to form spores in BHI and Columbia media was identified (Student's t test; P = 0.018; n = 4). The error bars indicate one standard deviation.

Effect of medium composition on spore germination with and without the cogerminant sodium taurocholate. The abilities of spores of eight clinical isolates of C. difficile to germinate on the corresponding agar form of the medium in the presence and absence of 1% sodium taurocholate were determined. The percentage of spore germination was calculated by dividing the number of spores germinated without taurocholate in the medium by the number of spores germinated with taurocholate in the medium. This value was then multiplied by 100 to obtain the percent germination. Each result is the mean of four repeats. CB, Columbia broth.

Relative hydrophobicities of spores formed by different ribotypes of C. difficile. The relative hydrophobicities of 21 clinical isolates of C. difficile belonging to different ribotypes were determined using a microbial adhesion to hydrocarbon test in which hexadecane was the organic solvent. Each value is the mean of two independent tests. The error bars indicate one standard deviation. Spores of B. atrophaeus ATCC 9372 were included as an internal control.

Adherence of C. difficile spores to human gut epithelial cell monolayers. The spores of DS1813 (hydrophobic) and DS1748 (hydrophilic) were assessed for the ability to adhere to monolayers of the human colon adenocarcinoma grade II cell line HT-29 and the Caco-2 human colorectal cell line that had been incubated for 7 and 15 days. Spores were enumerated on 0.1% sodium taurocholate-supplemented BHI agar. The results are presented as percentages of the initial spore count. Each result represents the mean of two independent assays. D, days.

Transmission electron microscopy of C. difficile spores. (A) TEM image of a cross section of a hydrophobic spore produced by C. difficile DS1813. The exosporial layer can be seen. (B) Negative-stain electron microscopy of an exosporium sac surrounding a spore of DS1813. (C) DS1813 with exosporium visible. Subsequent strains did not exhibit an exosporial layer. (D) Strain DS1771; the concentric layers of the spore can be seen, but no exosporial layer is visible. (E) Strain DS1684 does not exhibit an exosporium layer. (F) Strain DS1748 exhibits the concentric spore structure but does not appear to possess an exosporium. Scale bars = 100 nm.